Atherosclerosis with its sequelae of heart attack, strokes, and peripheral vascular disease is the leading cause of death in the United States with over 800,000 deaths per year. Excluding accidents, suicides, and homicides, atherosclerotic-related diseases account for nearly 50% of all deaths. Epidemiologic studies show that a large percentage of those afflicted have an elevation in blood low density lipoprotein (LDL) levels. LDL carries cholesterol from the liver to body tissues. An elevated cholesterol level (hypercholesterolemia) is commonly associated with an elevation in LDL levels. High blood cholesterol levels, specifically LDL-cholesterol, increase risk for coronary heart disease (CHD), whereas lowering total cholesterol and LDL-cholesterol levels reduces CHD risk.
Many pharmaceutical agents have been developed to treat or prevent atherosclerosis and its complications by controlling abnormally high blood LDL levels or lowering cholesterol levels. The most widely known of these agents include nicotinic acid, clofibrate, dextrothyroxine sodium, neomycin, beta-sitosterol, probucol, cholestyramine and HMG-CoA reductase inhibitors, such as lovastatin and simvastatin. However, the usefulness of these agents is limited by the frequent occurrence of acute side effects. Such side effects may include intense cutaneous flush, pruritus, gastrointestinal irritation, hepatotoxicity, cardiac arrhythmias, nausea, weight gain, alopecia, impotence, abdominal pain, diarrhea, eosinophilia, skin rash, musculoskeletal pain, blurred vision, mild anemia, leukopenia, the enhancement of gallstones, constipation, and impaction. Moreover, there is only a partial correlation between lowering of serum cholesterol and the reduction of atherosclerosis. Not all patients with atherosclerotic disease have high cholesterol and not all patients with high cholesterol have atherosclerotic disease.
The pathobiology of atherosclerosis indicates a major role for vascular endothelial involvement. Perturbation of the endothelium, without overt death and loss of endothelial cells, with resultant change in endothelial permeability to various blood materials is an important feature in the development of atherosclerotic lesions. Materials contained in the blood subsequently pass through this endothelial tissue and accumulate in the intima of the arterial wall. Even a moderate increase in endothelial permeability (hyperpermeability) is accompanied by a significant increase in the incidence of atherosclerotic events.
One mechanism by which vascular hyperpermeability can occur in the presence of an intact endothelium is increased endothelial endocytosis due to perturbation of the endothelium. The endothelium can be perturbed by various conditions, including high levels of low density lipoprotein (LDL) in the bloodstream and shear stress, as occurs in hypertension. Diabetes mellitus and smoking can also give rise to perturbation of the endothelium. In studies in support of the instant invention, human vascular endothelial cells (EC) were exposed to high LDL concentrations (up to 330 mg/dL cholesterol) for prolonged periods. The results, as demonstrated by stability of cell count for instance, indicate that EC death and loss do not occur in humans during the promotion of atherosclerotic plaque formation by LDL. When endothelial hyperpermeability is referred to herein, the process is primarily one of increased transcytosis rather than loss of physical integrity of the cell membrane or separation between formerly adherent cells.
Initially, endothelial perturbation occurs, resulting in increased endocytosis and LDL accumulation in the subendothelial space. Thus, exposure to high LDL levels induces heightened EC endocytosis. Studies indicate that exposure to LDL, in concentration ranges that are considered from epidemiologic studies to be atherogenic (endothelial cells exposed to &gt;140 mg/dL LDL cholesterol, for example), is necessary for exaggerated endocytosis. Another key finding is that once heightened endocytosis develops, it persists. Such a persistent change in EC functional state is consistent with the endothelial perturbation, or vascular hyperpermeability, concept.
Endocytosis is a fundamental, apparently ubiquitous, cellular event. During endocytosis, a segment of plasma membrane is interiorized to form a vesicle that migrates into the cytoplasm. This vesicle may participate in transcellular transport via transcytosis. The endocytosis regulatory mechanism is not well understood, but studies related to the instant invention demonstrate that reactive oxygen species, such as H.sub.2 O.sub.2 and O.sub.2.sup.-, enhance EC endocytosis. Cells generate reactive oxygen species (ROS) as byproducts of normal cellular metabolism. Perturbed endothelial cells increase reactive oxygen species production via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.
NADPH oxidase is an enzyme that has been well characterized in leukocytes, but leukocyte-type NADPH oxidase has not been previously identified in endothelial cells. Studies undertaken in support of the instant application have demonstrated that it occurs in endothelial cells. Activation of NADPH oxidase results in the transfer of electrons from NADPH to oxygen, resulting in the generation of O.sub.2-. The active form of the oxidase is a complex assembled from membrane-bound proteins that include cytochrome b.sub.558 (composed of gp91.sub.phox! and p22.sub.phox!) and cytosolic components, of which three have been characterized, p47.sub.phox!, p67.sub.phox! and a low molecular weight GTP-binding protein. Cytochrome b.sub.558 consists of a 22 kD polypeptide which tightly binds to a glycosylated 91 kD polypeptide.
It is known that inheritable abnormalities of the NADPH oxidase enzyme complex result in Chronic Granulomatous Disease (CGD), a congenital disorder in which leukocytes are unable to generate reactive oxygen species in response to microorganisms. Studies on CGD, in which NADPH oxidase activity is insufficient or absent, have shown that genetic variants of NADPH oxidase exist. There have been no studies, though, to identify NADPH oxidase genetic variants that produce excessive amounts of reactive oxygen species. If such variants exist and are common in our population, then people inheriting these genetic variants may be at higher risk of heart attack, stroke, and peripheral vascular disease.
The membrane-bound enzyme, NADPH oxidase, exists in an inactive form in quiescent cells. Upon cell perturbation, the enzyme complex assembles and is converted into an active state causing intensified reactive oxygen species generation (oxidative burst). Studies attempting to delineate the NADPH oxidase activation mechanism indicate that unsaturated fatty acids, most potently arachidonic acid, directly activate NADPH oxidase. To ascertain if arachidonic acid activates the NADPH oxidase found in endothelial cells, studies were carried out in which cells were directly exposed to increasing arachidonic acid concentrations and reactive oxygen species production was measured. For these studies, EC were incubated with 1 to 25 .mu.M arachidonic acid and H.sub.2 O.sub.2 production measured. It was shown that arachidonic acid both induces EC H.sub.2 O.sub.2 generation and promotes heightened EC endocytosis.
One explanation for the correlation between high LDL and atherosclerosis based on the above information is that exposure to high levels of LDL's promote phospholipase A.sub.2 activation. At an intracellular threshold level, cytosolic free arachidonic acid converts NADPH oxidase from a dormant to an active state.
Evidence from experiments conducted in support of the instant invention indicates that reactive oxygen species enhance endocytosis, a characteristic of atherosclerosis, and that NADPH oxidase is the major cellular source. Although NADPH oxidase inhibitors are claimed to be effective in treating inflammatory conditions, they have not been heretofore suggested to treat diseases such as atherosclerosis, whose initial phase is characterized by increased endocytosis and vascular hyperpermeability. For example, European patent application 551662 discloses the use of NADPH oxidase inhibitors to control acute and chronic inflammations of the airways, joints, and blood vessels. Such inflammations of the vessels include those arterioscleroses that are of inflammatory origin, but the EP application does not envision atherosclerosis, because the etiology of atherosclerosis (i.e., whether inflammatory or not) has not been established.
The use of 4-hydroxy-3-methoxyacetophenone (trivial name, apocynin) as an NADPH oxidase inhibitor is known, and it has been suggested to be of utility in treating inflammatory diseases. Apocynin is a natural phenol isolated from the root of the plant Picrorhiza kurroa, which grows in the Himalaya mountains. Extracts of Picrorhiza kurroa have been used in traditional medicine in Southeast Asia for the treatment of diseases connected with inflammation and for the treatment of a variety of conditions including liver and lung diseases, fever, skin lesions, worm infections, rheumatic disease, urinary disorders, heart failure, and snake and scorpion bites. A more recent reference Engels et al. FEBS Lett., 305, 254-56 (1992)! suggests that apocynin may also be useful in preventing thrombosis. However, neither apocynin nor any other NADPH oxidase inhibitor has been shown to prevent atherosclerosis or vascular hyperpermeability attributable to heightened EC endocytosis and high LDL concentrations.
A method that prevents and treats atherosclerosis and its associated diseases, that is effective in most patients, and that avoids patient-deterring side effects would represent a substantial advance toward eliminating a major cause of death in this country. A method employing a readily available medicament that has already been used in humans would present additional significant advantages in the prevention and treatment of atherosclerosis and related diseases. In addition, a diagnostic method for predicting an individual's potential risk of developing atherosclerosis is highly desirable.